(621c) A Systematic Analysis of Peptide Linker Length and Polyethylene Glycol Coating On Cellular Uptake of Peptide-Targeted Liposomes

Polyethylene glycol (PEG) coated liposomes with defined size range of 100 to 200 nm are widely used vehicles for the selective delivery of chemotherapeutics, most notably for cancer therapy. Liposomal drug formulations benefit from surface-grafted PEG by demonstrating increased stability, bioavailability, and tumor accumulation, while reducing systemic toxicity relative to small molecule drugs. These nanoparticles innately home to tumor sites via the enhanced penetration and retention (EPR) effect through a passive mechanism which can be further improved through active targeting. In an effort to improve tumor targeting and cellular uptake of nanoparticle-based drug carriers, nanoparticles are functionalized with active targeting molecules such as antibodies, antibody fragments, small molecules, and peptides. However, active targeting approaches have not consistently shown successful outcomes, largely due to the wide variety of tumor models employed with variable receptor targets, cellular growth rates, and uptake mechanisms. Additionally, nanoparticle design factors including: traditional methods of generating active targeting nanoparticles, PEG coatings of the particles, linkers used to conjugate the targeting ligands, and the various types of targeting ligands used further contribute to the observed targeting discrepancies.

On liposomal nanoparticles, PEG serves two functions: i) to provide stealth to the particles for increased circulation time and ii) to act as a linker to connect the targeting ligand to the particle. The current standard for establishing optimal in vivo circulation enhancement is the incorporation of a 5 mole percent methoxy-PEG2000-DSPE (PEG2000; a mean of ~45 repeating units of ethylene glycol: EG45) in the liposome formulation. This polymer length and percentage provides complete PEG coating of the liposome surface while maintaining particle stability. In order to create ligand-targeted nanoparticles, targeting ligands have been traditionally grafted onto the distal end of PEG2000. More recently, in an effort to more effectively present ligands above the PEG coating, extended linkers such as PEG3350 and PEG5000 have been successfully implemented. However, long PEG polymers do not preserve a linear conformation in the aqueous phase; instead, they fold within themselves to form globular structures. This unique morphology can bury a large fraction of conjugated ligand and sterically hinder the association of the ligand-targeted nanoparticles with their target receptor. Although PEG itself has been determined to be an ideal molecule to enhance bioavailability, the use of PEG2000 specifically as a liposomal coating and linker has been, in part, due to traditional reasons rather than scientific reasoning. Several reports in literature demonstrate that similar bioavailability profiles and in vivo circulation half-life can be achieved with much shorter liposomal PEG molecules such as PEG350, PEG550, and PEG750, despite the estimated PEG coverage of <100%. Furthermore, a shorter peptide linker may provide a more favorable thermodynamic receptor-ligand interaction by reducing entropic losses associated with long linkers. These provide a strong scientific rationale to evaluate the effect of shorter peptide linker lengths, in coordination with various liposomal PEG coatings, on tumor cell targeting and uptake.

Here, we employed a multifaceted synthetic strategy to prepare peptide-targeted liposomal nanoparticles with high purity, reproducibility, and precisely controlled stoichiometry of functionalities to evaluate the role of liposomal PEG coating, peptide EG-linker length, and peptide valency on cellular uptake in a systematic manner. We analyzed these parameters in two distinct disease models where the liposomes were functionalized with either HER2- or VLA-4-antagonistic peptides to target HER2-overexpressing breast cancer cells or VLA-4-overexpressing myeloma cells, respectively. When targeting peptides were tethered to nanoparticles with an EG45 (~PEG2000) linker in a manner similar to a more traditional formulation, their cellular uptake was not enhanced compared to non-targeted versions regardless of the liposomal PEG coating used. Conversely, reduction of the liposomal PEG to PEG350 and the peptide linker to EG12 dramatically enhanced cellular uptake by ~9-fold and ~100-fold in breast cancer and multiple myeloma cells, respectively. Uptake efficiency is highly dependent on peptide valency, reaching a maximum and a plateau with ~2% peptide density in both disease models. Taken together, these results demonstrate the significance of using the right design elements such as the appropriate peptide EG-linker length in coordination with the appropriate liposomal PEG coating and optimal ligand density in efficient cellular uptake of liposomal nanoparticles.